Homocysteine thiolactone is a cyclic thioester that is implicated in atherogenesis. This molecule will readily acylate primary amines, forming a homocystamide adduct, which contains a primary amine and a thiol. Here, we have characterized and evaluated the antioxidant potential of the homocystamide-low-density lipoprotein (LDL) adduct, a product of the acylation reaction between homocysteine thiolactone and LDL. Treatment of LDL with homocysteine thiolactone resulted in a time-dependent increase in LDL-bound thiol content that reached approximately 250 nmol thiol/mg LDL protein at 75 minutes. The increase in LDL thiol content was followed by aggregation of LDL after 75 minutes. The increase in LDL-bound thiols was reversible by treatment with the thiol blockersing spin label, methanethiosulfonate. As assessed by the electron spin resonance (ESR) spin labeling technique, the homocystamide adducts were predominately exposed to the aqueous phase of LDL, shown by the sharp ESR spectra that were greatly broadened by the hydrophillic paramagnetic relaxing agent, chromium oxalate. The relative electrophoretic mobility of the homocystamide-LDL adduct was increased with respect to native LDL. Primary amino group concentration of homocysteine thiolactone-treated LDL was not significantly different than native LDL (p < 0.05). The homocystamide-LDL adduct was resistant to Cu(2+)- and 2,2'-azobis (2-amidinopropane)- mediated oxidation (with respect to native LDL) as measured by the formation of thiobarbituric reactive substances and the depletion of vitamin E. Blocking thiols with N-ethylmaleamide prevented the antioxidant effect of the homocystamide-LDL adduct. The potential relationship between the homocystamide-LDL adduct and the development of atherosclerosis is discussed.